Methods and compositions for treating and diagnosing kidney disease

ABSTRACT

The invention relates to a method for diagnosing a kidney disease state. The method comprises the steps of administering to a patient a composition comprising a conjugate or complex of the general formula V-L-D where the group V comprises a vitamin receptor binding ligand that binds to kidney proximal tubule cells and the group D comprises a diagnostic marker, and diagnosing the kidney disease state. The invention also relates to a method for treating a kidney disease state. The method comprises the steps of administering to a patient suffering from the disease state an effective amount of a composition comprising a conjugate or complex of the general formula V-L-D where the group V comprises a vitamin receptor binding ligand that binds to kidney proximal tubule cells and the group D comprises an antigen, a cytotoxin, or a cell growth inhibitor, and eliminating the disease state.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 60/901,778, filed on Feb. 16, 2007, the entire disclosure ofwhich is hereby incorporated by reference.

FIELD OF THE INVENTION

This invention relates to methods and compositions for treating anddiagnosing kidney disease states. More particularly, ligands that bindto receptors overexpressed on proximal tubule cells are complexed with adiagnostic marker for use in diagnosis or to an antigen, a cytotoxin, ora cell growth inhibitor for use in the treatment of kidney diseasestates.

BACKGROUND

Diseases affecting kidney function are prevalent. For example,polycystic kidney disease (PKD) is a prevalent inherited disease. AdultPKD is an autosomal dominant disorder affecting approximately 600,000people in the United States and 12.5 million world-wide. Infants canalso present with autosomal recessive PKD which is rapidly developingand which can lead to renal insufficiency in the neonate. PKD and otherkidney disease states (e.g., Dent's disease and nephrocytinosis) affectand manifest abnormal growth of kidney proximal tubule cells. PKDresults in the proliferation of kidney epithelial cells and theformation of PKD renal cysts. The kidneys can become enlarged andsymptoms including pain, bleeding, and kidney stones can occur.Associated problems include liver cysts, abdominal aneurysm,intracranial aneurysm, and renal insufficiency. It has been suggestedthat cellular processes associated with signal transduction,transcriptional regulation, and cell-cycle control are involved in cystformation in PKD.

The folate receptor is a 38 KD GPI-anchored protein that binds thevitamin folic acid with high affinity (<1 nM). Following receptorbinding, rapid endocytosis delivers a substantial fraction of thevitamins into the cell, where they are unloaded in an endosomalcompartment at low pH. Importantly, covalent conjugation of smallmolecules, proteins, and even liposomes to folic acid does not block thevitamin's ability to bind the folate receptor, and therefore,folate-drug conjugates can readily be delivered to and can enter cellsby receptor-mediated endocytosis. Because most cells use an unrelatedreduced folate carrier to acquire the necessary folic acid, expressionof the folate receptor is restricted to a few cell types, and normaltissues typically express low or nondetectable levels of the folatereceptor. Folate receptors are overexpressed in proximal tubule cells.

The invention is based on the manifestation of abnormal proliferation ofkidney proximal tubule cells in PKD and other kidney disease states thatexhibit abnormal proximal tubule cell proliferation. These kidneydisease states can be treated with ligands that bind to receptorsoverexpressed on proximal tubule cells wherein the ligands are complexedwith an antigen, a cytotoxin, or a cell growth inhibitor for use in thetreatment of the kidney disease states. These kidney disease states,including PKD, can also be diagnosed by using ligands that bind toreceptors overexpressed on proximal tubule cells wherein the ligands arecomplexed with a diagnostic marker.

SUMMARY

In one embodiment, a method for diagnosing a kidney disease state isprovided. The method comprises the steps of administering to a patient acomposition comprising a conjugate or complex of the general formulaV-L-D, where the group V comprises a vitamin receptor binding ligandthat binds to kidney cells and the group D comprises a diagnosticmarker, and diagnosing the kidney disease state.

In another embodiment, V comprises a folate receptor binding ligand or Vcomprises a folate receptor binding antibody or antibody fragment. Inyet another embodiment, the marker can comprise a metal chelatingmoiety, or a fluorescent chromophore. In another illustrativeembodiment, the disease state is selected from the group consisting ofpolycystic kidney disease, Dent's disease, nephrocytinosis, and Heymannnephritis.

In another embodiment, a method for treating a kidney disease state isprovided. The method comprises the steps of administering to a patientsuffering from the disease state an effective amount of a compositioncomprising a conjugate or complex of the general formula V-L-D where thegroup V comprises a vitamin receptor binding ligand that binds to kidneycells and the group D comprises an antigen, a cytotoxin, or a cellgrowth inhibitor, and eliminating the disease state.

In another embodiment, V comprises a folate receptor binding ligand oran antibody or antibody fragment that binds to the folate receptor. Inanother illustrative aspect, group D comprises an antigen, a cytotoxin,or a cell growth inhibitor. In yet another embodiment, the cell growthinhibitor is selected from the group consisting of epidermal growthfactor receptor kinase inhibitors, inhibitors of the mTOR pathway, DNAalkylators, microtubule inhibitors, cell cycle inhibitors, and proteinsynthesis inhibitors. In another embodiment, the disease state isselected from the group consisting of polycystic kidney disease, Dent'sdisease, nephrocytinosis, and Heymann nephritis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows IHC analysis of folate receptor expression in polycystickidney disease tissues using a monoclonal antibody directed to thefolate receptor for staining. The upper left panel shows normal humankidney tissue and the remainder of the panels show staining of cysts inpolycystic kidney disease tissues using the anti-folate receptormonoclonal antibody.

FIG. 2 shows IHC analysis of folate receptor expression in polycystickidney disease tissues using a polyclonal antibody directed to thefolate receptor for staining. The upper left panel shows normal mousekidney tissue and the remainder of the panels show staining of cysts inpolycystic kidney disease tissues using the anti-folate receptorpolyclonal antibody.

FIG. 3 shows the structure of EC0371, a folate-rapamycin conjugate.

FIG. 4 shows an affinity assay comparing the relative affinities offolic acid (circles; 1.0) and EC0371 (triangles; 0.5) for the folatereceptor.

FIG. 5 shows the effect of rapamycin and EC0371 on the viability of KBcells at various free rapamycin and conjugated rapamycin (EC0371)concentrations. The leftmost panels show untreated cells. The panels inthe second column from the left show control cells treated with DMSO(diluent). The panels in the third column from the left show cellstreated with 2, 10, or 50 nM rapamycin. The panels in the rightmostcolumn show cells treated with 2, 10, or 50 nM EC0371. Neither rapamycinnor EC0371 is cytotoxic after 24 hours of treatment.

FIG. 6 shows the effects of rapamycin and EC0371 on P-S6 immunostainingin KB cells after 16 hours of incubation with rapamycin or EC0371. P-S6is a phosphorylation target of m-TOR and the antibody used isphospho-specific. The leftmost panels show untreated cells. The panelsin the middle column show cells treated with 2, 10, or 50 nM rapamycin.The panels in the rightmost column show cells treated with 2, 10, or 50nM EC0371. Rapamycin and EC0371 inhibit P-S6 immunostaining (i.e.,phosphorylation of P-S6 through the mTOR pathway).

FIG. 7 shows an immunoblot using a phospho-specific antibody. The leftpanel shows phosphorylation of ribosomal S6 and S-6 kinase (T389) inuntreated cells and cells treated with DMSO (diluent). The right panelshows that rapamycin (2, 10, and 50 nM) and EC0371 (folate-rapamyin; 2,10, and 50 nM) abolish or greatly reduce phosphorylation of ribosomal S6and S-6 kinase (T389) which are phosphorylation targets in the m-TORpathway.

FIG. 8 shows the therapeutic effect of EC0371 on the in vivo developmentof polycystic kidney disease in the bpk-mutant mouse model. The leftmostkidney is from a wildtype mouse. The middle kidney is from a bpk mutantmouse not treated with EC0371. The rightmost kidney is from a bpk mutantmouse treated with EC0371 showing that EC0371 greatly reduces kidneysize.

FIG. 9 shows the effect on one-kidney weight of EC0371 treatment inmultiple bpk mutant mice (rightmost group of symbols). EC0371-treatedbpk mice exhibit a significant decrease in one-kidney weight as apercentage of total body weight relative to untreated bpk mice.

FIG. 10 shows the effect on two-kidney weight of EC0371 treatment inmultiple bpk mutant mice (rightmost group of symbols). EC0371-treatedbpk mice exhibit a significant decrease in two-kidney weight as apercentage of total body weight relative to untreated bpk mice.

DETAILED DESCRIPTION

Methods are provided for treating and diagnosing kidney disease states.Exemplary disease states include PKD, Dent's disease, nephrocytinosis,Heymann nephritis, and other diseases manifested by abnormalproliferation of proximal tubule cells of the kidney. PKD's can include,but are not limited to, autosomal dominant (adult) polycystic kidneydisease and autosomal recessive (childhood) polycystic kidney disease.These disease states are characterized by abnormal proliferation ofkidney proximal tubule cells. Such disease states can be diagnosed bycontacting kidney proximal tubule cells with a composition comprising aconjugate of the general formula V-L-D wherein the group V comprises aligand that binds to the kidney proximal tubule cells, and the group Dcomprises a diagnostic marker, and diagnosing the disease state. Suchdisease states can be treated by contacting kidney proximal tubule cellswith a composition comprising a conjugate of the general formula V-L-Dwherein the group V comprises a ligand that binds to the kidney proximaltubule cells, and the group D comprises an antigen, a cytotoxin, or acell growth inhibitor, and eliminating the disease state.

As used herein, the terms “eliminated” and “eliminating” in reference tothe disease state, mean reducing the symptoms or eliminating thesymptoms of the disease state or preventing the progression or thereoccurrence of disease.

As used herein, the term “elimination” of the proximal tubule cellpopulation causing the disease state that expresses the ligand receptormeans that this cell population is killed or is completely or partiallyremoved or inactivated which reduces the pathogenic characteristics ofthe disease state being treated.

The kidney disease states characterized by abnormal proliferation ofproximal tubule cells can be treated in accordance with the methodsdisclosed herein by administering an effective amount of a compositionV-L-D wherein V comprises a ligand that binds to proximal tubule cellsand wherein the group D comprises an antigen, a cytotoxin, or a cellgrowth inhibitor. Such targeting conjugates, when administered to apatient suffering from a kidney disease state manifested by abnormalproximal tubule cell proliferation, work to concentrate and associatethe conjugated cytotoxin, antigen, or cell growth inhibitor with thepopulation of proximal tubule cells to kill the cells or alter cellfunction. The conjugate is typically administered parenterally, but canbe delivered by any suitable method of administration (e.g., orally), asa composition comprising the conjugate and a pharmaceutically acceptablecarrier therefor. Conjugate administration is typically continued untilsymptoms of the disease state are reduced or eliminated, oradministration is continued after this time to prevent progression orreappearance of the disease.

For diagnosis the typical method of administration of the conjugates isparenteral administration, but any suitable method can be used. In thisembodiment, kidney disease states can be diagnosed by administeringparenterally to a patient a composition comprising a conjugate orcomplex of the general formula V-L-D where the group V comprises aligand that binds to proximal tubule cells and the group D comprises adiagnostic marker, and diagnosing the disease state.

In one embodiment, for example, the diagnostic marker (e.g., a reportermolecule) can comprise a radiolabeled compound such as a chelatingmoiety and an element that is a radionuclide, for example a metal cationthat is a radionuclide. In another embodiment, the radionuclide isselected from the group consisting of technetium, gallium, indium, and apositron emitting radionuclide (PET imaging agent). In anotherembodiment, the diagnostic marker can comprise a fluorescent chromophoresuch as, for example, fluorescein, rhodamine, Texas Red, phycoerythrin,Oregon Green, AlexaFluor 488 (Molecular Probes, Eugene, Oreg.), Cy3,Cy5, Cy7, and the like. Imaging agents are described in U.S. Pat. No.7,128,893 and in U.S. Patent Publ. No. 20070009434, each incorporatedherein by reference.

Diagnosis typically occurs before treatment. However, in the diagnosticmethods described herein, the term “diagnosis” can also mean monitoringof the disease state before, during, or after treatment to determine theprogression of the disease state. The monitoring can occur before,during, or after treatment, or combinations thereof, to determine theefficacy of therapy, or to predict future episodes of disease. Thediagnostic method can be any suitable method known in the art, includingimaging methods, such as intravital imaging.

The method disclosed herein can be used for both human clinical medicineand veterinary applications. Thus, the patient or animal afflicted withthe kidney disease state and in need of diagnosis or therapy can be ahuman, or in the case of veterinary applications, can be a laboratory,agricultural, domestic or wild animal. In embodiments where theconjugates are administered to the patient or animal, the conjugates canbe administered parenterally to the animal or patient suffering from thekidney disease state, for example, intradermally, subcutaneously,intramuscularly, intraperitoneally, or intravenously. Alternatively, theconjugates can be administered to the animal or patient by othermedically useful procedures and effective doses can be administered instandard or prolonged release dosage forms, such as a slow pump. Thetherapeutic method described herein can be used alone or in combinationwith other therapeutic methods recognized for the treatment of kidneydisease states.

In the ligand conjugates of the general formula V-L-D, the group V is aligand that binds to proximal tubule cells when the conjugates are usedto diagnose or treat kidney disease states. Any of a wide number ofbinding ligands can be employed. Acceptable ligands include, forexample, folate receptor binding ligands, and analogs thereof, andantibodies or antibody fragments capable of recognizing and binding tosurface moieties expressed on proximal tubule cells, in particular whenthese cells proliferate abnormally. In one embodiment, the bindingligand is folic acid, a folic acid analog, or another folate receptorbinding molecule. In another embodiment the binding ligand is a specificmonoclonal or polyclonal antibody or an Fab or an scFv (i.e., a singlechain variable region) fragment of an antibody capable of binding toreceptors overexpressed on proximal tubule cells, for example, whenthese cells proliferate abnormally.

In one embodiment, the binding ligand can be folic acid, a folic acidanalog, or another folate receptor-binding molecule. Analogs of folatethat can be used include folinic acid, pteropolyglutamic acid, andfolate receptor-binding pteridines such as tetrahydropterins,dihydrofolates, tetrahydrofolates, and their deaza and dideaza analogs.The terms “deaza” and “dideaza” analogs refers to the art recognizedanalogs having a carbon atom substituted for one or two nitrogen atomsin the naturally occurring folic acid structure. For example, the deazaanalogs include the 1-deaza, 3-deaza, 5-deaza, 8-deaza, and 10-deazaanalogs. The dideaza analogs include, for example, 1,5 dideaza,5,10-dideaza, 8,10-dideaza, and 5,8-dideaza analogs. The foregoing folicacid analogs are conventionally termed “folates,” reflecting theircapacity to bind to folate receptors. Other folate receptor-bindinganalogs include aminopterin, amethopterin (methotrexate),N¹⁰-methylfolate, 2-deamino-hydroxyfolate, deaza analogs such as1-deazamethopterin or 3-deazamethopterin, and3′,5′-dichloro-4-amino-4-deoxy-N¹⁰-methylpteroylglutamic acid(dichloromethotrexate).

In another embodiment, other vitamins can be used as the binding ligand.The vitamins that can be used in accordance with the methods describedherein include niacin, pantothenic acid, folic acid, riboflavin,thiamine, biotin, vitamin B₁₂, vitamins A, D, E and K, other relatedvitamin molecules, analogs and derivatives thereof, and combinationsthereof.

In other embodiments, the binding ligand can be any ligand that binds toa receptor expressed or overexpressed on proximal tubule cells, inparticular when they proliferate abnormally (e.g., EGF, KGF, or leptin).In another embodiment, the binding ligand can be any ligand that bindsto a receptor expressed or overexpressed on proximal tubule cellsproliferating abnormally and involved in a kidney disease state.

The targeted conjugates used for diagnosing or treating disease statesmediated by proximal tubule cells proliferating abnormally have theformula V-L-D, wherein V is a ligand capable of binding to the proximaltubule cells, and the group D comprises a diagnostic marker or anantigen (such as an immunogen), cytotoxin, or a cell growth inhibitor.In such conjugates wherein the group V is folic acid, a folic acidanalog, or another folic acid receptor binding ligand, these conjugatesare described in detail in U.S. Pat. No. 5,688,488, the specification ofwhich is incorporated herein by reference. That patent, as well asrelated U.S. Pat. Nos. 5,416,016 and 5,108,921, and related U.S. patentapplication Ser. No. 10/765,336, each incorporated herein by reference,describe methods and examples for preparing conjugates useful inaccordance with the methods described herein. The present targeteddiagnostic and therapeutic agents can be prepared and used followinggeneral protocols described in those earlier patents and patentapplications, and by the protocols described herein.

In accordance with another embodiment, there is provided a method oftreating kidney disease states by administering to a patient sufferingfrom such disease state an effective amount of a composition comprisinga conjugate of the general formula V-L-D wherein V is as defined aboveand the group D comprises a cytotoxin, an antigen (i.e., a compoundadministered to a patient for the purpose of eliciting an immuneresponse in vivo), or a cell growth inhibitor. The group V can be any ofthe ligands described above. Exemplary of cytotoxic moieties useful forforming conjugates for use in accordance with the methods describedherein include art-recognized chemotherapeutic agents such asantimetabolites, methotrexate, busulfan, carboplatin, chlorambucil,cisplatin and other platinum compounds, plant alkaloids, hydroxyurea,teniposide, and bleomycin, MEK kinase inhibitors, MAP kinase pathwayinhibitors, PI-3-kinase inhibitors, NFκB pathway inhibitors,pro-apoptotic agents, apoptosis-inducing agents, proteins such aspokeweed, saporin, momordin, and gelonin, didemnin B, verrucarin A,geldanamycin, toxins, and the like. Such cytotoxic compounds can bedirectly conjugated to the targeting ligand, for example, folate oranother folate receptor-binding ligand, or they can be formulated inliposomes or other small particles which themselves can be targeted toproximal tubule cells by pendent targeting ligands V non-covalently orcovalently linked to one or more liposome components.

In another embodiment, the group D comprises a cell growth inhibitor,and the inhibitor can be covalently linked to the targeting ligand V,for example, a folate receptor-binding ligand or a proximal tubulecell-binding antibody or antibody fragment (i.e., an antibody to areceptor overexpressed on proximal tubule cells that are proliferatingabnormally). The ligand can be linked directly, or the ligand can beencapsulated in a liposome which is itself targeted to the proximaltubule cells by pendent targeting ligands V covalently or non-covalentlylinked to one or more liposome components. Cell growth inhibitors can beselected from the group consisting of epidermal growth factor receptorkinase inhibitors and other kinase inhibitors (e.g. rapamycin and otherinhibitors of the mTOR pathway, r-roscovitine and other cyclin-dependentkinase inhibitors), DNA alkylators (e.g., nitrogen mustards (e.g.,cyclophosphamide), ethyleneamines, alkyl sulfonates, nitrosoureas, andtriazene derivatives), microtubule inhibitors (e.g., tamoxiphen,paclitaxel, docetaxel (and other taxols), vincristine, vinblastine,colcemid, and colchicine), cell cycle inhibitors (e.g., cytosinearabinoside, purine analogs, and pyrimidine analogs), and proteinsynthesis inhibitors (e.g., proteosome inhibitors). In one embodiment,rapamycin (RAPAMUNE®, Wyeth Pharmaceuticals, Inc., Madison, N.J.) is thecell growth inhibitor. Rapamycin is described in Shillingford, et al.,PNAS 103: 5466-5471 (2006), incorporated herein by reference. In anotherembodiment, more than one of these drugs can be conjugated to a ligand,such as folate, to form, for example, a dual-drug conjugate.

In another embodiment, conjugates V-L-D where D is an antigen or a cellgrowth inhibitor can be administered in combination with a cytotoxiccompound. The cytotoxic compounds listed above are among the compoundssuitable for this purpose.

In one embodiment, conjugates are described herein, and such conjugatesmay be used in the treatment methods described herein. Illustratively,the conjugates have the general formula

V-L-D

where V is a folate receptor binding ligand, L is an optional linker,and D is a cell-growth inhibitor, an antigen, or a cytotoxin.

In one embodiment, the folate receptor binding ligand is folate or ananalog of folate, or alternatively a derivative of either folate or ananalog thereof. As used herein, the term “folate” or “folates” may referto folate itself, or such analogs and derivatives of folate. However, itis to be understood that other folate receptor binding ligands inaddition to folates are contemplated herein. Illustratively, such folatereceptor binding ligands include any compound capable or specific orselective binding to folate receptors, especially those receptorspresent on the surface of cells.

In another embodiment, the optional linker is absent, and the conjugateis formed by directly attaching the folate receptor binding ligand tothe cell-growth inhibitor, a cytotoxin, or an antigen. In anotherembodiment, the optional linker is present and is a divalent chemicalfragment comprising a chain of carbon, nitrogen, oxygen, silicon,sulfur, and phosphorus. It is to be understood that the foregoing atomsmay be arranged in any chemically meaningful way. In one variation,peroxide bonds, i.e. —O—O— do not form part of the linker. Generally,the linker is formed from the foregoing atoms by arranging those atomsto form functional groups, including but not limited to, alkylene,cycloalkylene, arylene, ether, amino, hydroxylamino, oximino, hydrazine,hydrazono, thio, disulfide, carbonyl, carboxyl, carbamoyl, thiocarbonyl,thiocarboxyl, thiocarbamoyl, xanthyl, silyl, phosphinyl, phosphonyl,phosphate, and like groups that may be linked together to construct thelinker. It is appreciated that each of these fragments may also beindependently substituted.

In another embodiment, the drug is a cell-growth inhibitor. Illustrativeof such cell-growth inhibitors are epidermal growth factor (EGF)receptor kinase inhibitors. Further illustrative of such cell-growthinhibitor are DNA alkylators, microtubule inhibitors, cell cycleinhibitors, and protein synthesis inhibitors.

In another illustrative embodiment, such cell growth inhibitors arecompounds that inhibit the mammalian target of rapamycin, also referredto as mTOR. mTOR is a serine/threonine protein kinase that has beenreported to regulate cell growth, cell proliferation, cell motility,cell survival, protein synthesis, and transcription (see generally,Beevers et al. “Curcumin inhibits the mammalian target ofrapamycin-mediated signaling pathways in cancer cells,” InternationalJournal of Cancer, 119(4):757-64 (2006); Hay & Sonenberg N “Upstream anddownstream of mTOR,” Genes & Development, 18(16): 1926-45 (2004)). mTORhas been shown to function as the catalytic subunit of two distinctmolecular complexes in cells. mTOR Complex 1 (mTORC1) is composed ofmTOR, regulatory associated protein of mTOR (Raptor), and mammalianLST8/G-protein fl-subunit like protein (mLST8/GβL). This complexpossesses the classic features of mTOR by functioning as anutrient/energy/redox sensor and controlling protein synthesis. mTORComplex 2 (mTORC2) is composed of mTOR, rapamycin-insensitive companionof mTOR (Rictor), GβL, and mammalian stress-activated protein kinaseinteracting protein 1 (mSIN1). mTORC2 has been shown to function as animportant regulator of the cytoskeleton through its stimulation ofF-actin stress fibers, paxillin, RhoA, Rac1, Cdc42, and protein kinase Ca (PKCα). In addition, mTORC2 has also been reported to be a “PDK2.”

Illustrative of such mTOR inhibitors is rapamycin, and analogs andderivatives of rapamycin, such as are described in U.S. Pat. Nos.7,153,957 (Regioselective synthesis of CCI-779), 7,122,361 (Compositionsemploying a novel human kinase), 7,105,328 (Methods for screening forcompounds that modulate pd-1 signaling), 7,074,804 (CCI-779 Isomer C),7,060,797 (Composition and method for treating lupus nephritis),7,060,709 (Method of treating hepatic fibrosis), 7,029,674 (Methods fordownmodulating immune cells using an antibody to PD-1), 7,019,014(Process for producing anticancer agent LL-D45042), 6,958,153 (Skinpenetration enhancing components), 6,821,731 (Expression analysis ofFKBP nucleic acids and polypeptides useful in the diagnosis of prostatecancer), 6,713,607 (Effector proteins of Rapamycin), 6,680,330(Rapamycin dialdehydes), 6,677,357 (Rapamycin 29-enols), 6,670,355(Method of treating cardiovascular disease), 6,617,333 (Antineoplasticcombinations), 6,541,612 (Monoclonal antibodies obtained using rapamycinposition 27 conjugates as an immunogen), 6,511,986 (Method of treatingestrogen receptor positive carcinoma), 6,440,991 (Ethers of7-desmethylrapamycin), 6,432,973 (Water soluble rapamycin esters),6,399,626 (Hydroxyesters of 7-desmethylrapamycin), and 6,399,625(1-oxorapamycins), each incorporated herein by reference.

In another illustrative embodiment, the linker includes an amino acid ora peptide from 2 to about 20 amino acids in length. As used herein, itis to be understood that amino acids are illustratively selected fromthe naturally occurring amino acids, or stereoisomers thereof. Inaddition, amino acids may be non-naturally occurring, and have forexample the general formula:

—N(R)—(CR′R″)_(q)—C(O)—

where R is hydrogen, alkyl, acyl, or a suitable nitrogen protectinggroup, R′ and R″ are hydrogen or a substituent, each of which isindependently selected in each occurrence, and q is an integer such as1, 2, 3, 4, or 5. Illustratively, R′ and/or R″ independently correspondto, but are not limited to, hydrogen or the side chains present onnaturally occurring amino acids, such as methyl, benzyl, hydroxymethyl,thiomethyl, carboxyl, carboxylmethyl, guanidinopropyl, and the like, andderivatives and protected derivatives thereof. The above describedformula includes all stereoisomeric variations. It is furtherappreciated that water solubilizing amino acids may be included in thelinker to facilitate uptake and transport of the conjugates describedherein. For example, the amino acids may be selected from asparagine,aspartic acid, cysteine, glutamic acid, lysine, glutamine, arginine,serine, ornithine, threonine, and the like.

In another illustrative embodiment, the bivalent linker (L) comprisesone or more spacer linkers, heteroatom linkers, and releasable (i.e.,cleavable) linkers, and combinations thereof, in any order. The term“releasable linker” as used herein generally refers to a linker thatincludes at least one bond that can be broken under physiologicalconditions (e.g., a pH-labile, acid-labile, oxidatively-labile,enzyme-labile bond, and the like). It is appreciated that suchphysiological conditions resulting in bond breaking include standardchemical hydrolysis reactions that occur, for example, at physiologicalpH, or as a result of compartmentalization into a cellular organellesuch as an endosome having a lower pH than cytosolic pH.

It is also understood that a cleavable bond can connect two adjacentatoms within the releasable linker and/or connect other linkers or Vand/or D, as described herein, at either or both ends of the releasablelinker. In the case where a cleavable bond connects two adjacent atomswithin the releasable linker, following breakage of the bond, thereleasable linker is broken into two or more fragments. Alternatively,in the case where a cleavable bond is between the releasable linker andanother moiety, such as an heteroatom linker, a spacer linker, anotherreleasable linker, the drug, or analog or derivative thereof, or thevitamin, or analog or derivative thereof, following breakage of thebond, the releasable linker is separated from the other moiety.

The lability of the cleavable bond can be adjusted by, for example,substitutional changes at or near the cleavable bond, such as includingalpha branching adjacent to a cleavable disulfide bond, increasing thehydrophobicity of substituents on silicon in a moiety havingsilicon-oxygen bond that may be hydrolyzed, homologating alkoxy groupsthat form part of a ketal or acetal that may be hydrolyzed, and thelike.

In one embodiment, the present invention provides a vitamin receptorbinding drug delivery conjugate. The drug delivery conjugate consists ofa vitamin receptor binding moiety, bivalent linker (L), and a drug. Thevitamin receptor binding moiety is a vitamin, or an analog or aderivative thereof, capable of binding to vitamin receptors, and thedrug (antigen, cytotoxin, or cell growth inhibitor) includes analogs orderivatives thereof exhibiting drug activity. The vitamin, or the analogor the derivative thereof, is covalently attached to the bivalent linker(L), and the drug, or the analog or the derivative thereof, is alsocovalently attached to the bivalent linker (L). The bivalent linker (L)comprises one or more spacer linkers, releasable linkers, and heteroatomlinkers, and combinations thereof, in any order. For example, theheteroatom linker can be nitrogen, and the releasable linker and theheteroatom linker can be taken together to form a divalent radicalcomprising alkyleneaziridin-1-yl, alkylenecarbonylaziridin-1-yl,carbonylalkylaziridin-1-yl, alkylenesulfoxylaziridin-1-yl,sulfoxylalkylaziridin-1-yl, sulfonylalkylaziridin-1-yl, oralkylenesulfonylaziridin-1-yl, wherein each of the releasable linkers isoptionally substituted with a substituent X², as defined below.Alternatively, the heteroatom linkers can be nitrogen, oxygen, sulfur,and the formulae —(NHR¹NHR²)—, —SO—, —(SO₂)—, and —N(R³)O—, wherein R¹,R², and R³ are each independently selected from hydrogen, alkyl, aryl,arylalkyl, substituted aryl, substituted arylalkyl, heteroaryl,substituted heteroaryl, and alkoxyalkyl. In another embodiment, theheteroatom linker can be oxygen, the spacer linker can be1-alkylenesuccinimid-3-yl, optionally substituted with a substituent X¹,as defined below, and the releasable linkers can be methylene,1-alkoxyalkylene, 1-alkoxycycloalkylene, 1-alkoxyalkylenecarbonyl,1-alkoxycycloalkylenecarbonyl, wherein each of the releasable linkers isoptionally substituted with a substituent X², as defined below, andwherein the spacer linker and the releasable linker are each bonded tothe heteroatom linker to form a succinimid-1-ylalkyl acetal or ketal.

The spacer linkers can be carbonyl, thionocarbonyl, alkylene,cycloalkylene, alkylenecycloalkyl, alkylenecarbonyl,cycloalkylenecarbonyl, carbonylalkylcarbonyl, 1-alkylenesuccinimid-3-yl,1-(carbonylalkyl)succinimid-3-yl, alkylenesulfoxyl, sulfonylalkyl,alkylenesulfoxylalkyl, alkylenesulfonylalkyl,carbonyltetrahydro-2H-pyranyl, carbonyltetrahydrofuranyl,1-(carbonyltetrahydro-2H-pyranyl)succinimid-3-yl, and1-(carbonyltetrahydrofuranyl)succinimid-3-yl, wherein each of the spacerlinkers is optionally substituted with a substituent X¹, as definedbelow. In this embodiment, the heteroatom linker can be nitrogen, andthe spacer linkers can be alkylenecarbonyl, cycloalkylenecarbonyl,carbonylalkylcarbonyl, 1-(carbonylalkyl)succinimid-3-yl, wherein each ofthe spacer linkers is optionally substituted with a substituent X¹, asdefined below, and the spacer linker is bonded to the nitrogen to forman amide. Alternatively, the heteroatom linker can be sulfur, and thespacer linkers can be alkylene and cycloalkylene, wherein each of thespacer linkers is optionally substituted with carboxy, and the spacerlinker is bonded to the sulfur to form a thiol. In another embodiment,the heteroatom linker can be sulfur, and the spacer linkers can be1-alkylenesuccinimid-3-yl and 1-(carbonylalkyl)succinimid-3-yl, and thespacer linker is bonded to the sulfur to form a succinimid-3-ylthiol.

In an alternative to the above-described embodiments, the heteroatomlinker can be nitrogen, and the releasable linker and the heteroatomlinker can be taken together to form a divalent radical comprisingalkyleneaziridin-1-yl, carbonylalkylaziridin-1-yl,sulfoxylalkylaziridin-1-yl, or sulfonylalkylaziridin-1-yl, wherein eachof the releasable linkers is optionally substituted with a substituentX², as defined below. In this alternative embodiment, the spacer linkerscan be carbonyl, thionocarbonyl, alkylenecarbonyl,cycloalkylenecarbonyl, carbonylalkylcarbonyl,1-(carbonylalkyl)succinimid-3-yl, wherein each of the spacer linkers isoptionally substituted with a substituent X¹, as defined below, andwherein the spacer linker is bonded to the releasable linker to form anaziridine amide.

The substituents X¹ can be alkyl, alkoxy, alkoxyalkyl, hydroxy,hydroxyalkyl, amino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl,halo, haloalkyl, sulfhydrylalkyl, alkylthioalkyl, aryl, substitutedaryl, arylalkyl, substituted arylalkyl, heteroaryl, substitutedheteroaryl, carboxy, carboxyalkyl, alkyl carboxylate, alkyl alkanoate,guanidinoalkyl, R⁴-carbonyl, R⁵-carbonylalkyl, R⁶-acylamino, andR⁷-acylaminoalkyl, wherein R⁴ and R⁵ are each independently selectedfrom amino acids, amino acid derivatives, and peptides, and wherein R⁶and R⁷ are each independently selected from amino acids, amino acidderivatives, and peptides. In this embodiment the heteroatom linker canbe nitrogen, and the substituent X¹ and the heteroatom linker can betaken together with the spacer linker to which they are bound to form anheterocycle.

The releasable linkers can be methylene, 1-alkoxyalkylene,1-alkoxycycloalkylene, 1-alkoxyalkylenecarbonyl,1-alkoxycycloalkylenecarbonyl, carbonylarylcarbonyl,carbonyl(carboxyaryl)carbonyl, carbonyl(biscarboxyaryl)carbonyl,haloalkylenecarbonyl, alkylene(dialkylsilyl), alkylene(alkylarylsilyl),alkylene(diarylsilyl), (dialkylsilyl)aryl, (alkylarylsilyl)aryl,(diarylsilyl)aryl, oxycarbonyloxy, oxycarbonyloxyalkyl, sulfonyloxy,oxysulfonylalkyl, iminoalkylidenyl, carbonylalkylideniminyl,iminocycloalkylidenyl, carbonylcycloalkylideniminyl, alkylenethio,alkylenearylthio, and carbonylalkylthio, wherein each of the releasablelinkers is optionally substituted with a substituent X², as definedbelow.

In the preceding embodiment, the heteroatom linker can be oxygen, andthe releasable linkers can be methylene, 1-alkoxyalkylene,1-alkoxycycloalkylene, 1-alkoxyalkylenecarbonyl, and1-alkoxycycloalkylenecarbonyl, wherein each of the releasable linkers isoptionally substituted with a substituent X², as defined below, and thereleasable linker is bonded to the oxygen to form an acetal or ketal.Alternatively, the heteroatom linker can be oxygen, and the releasablelinker can be methylene, wherein the methylene is substituted with anoptionally-substituted aryl, and the releasable linker is bonded to theoxygen to form an acetal or ketal. Further, the heteroatom linker can beoxygen, and the releasable linker can be sulfonylalkyl, and thereleasable linker is bonded to the oxygen to form an alkylsulfonate.

In another embodiment of the above releasable linker embodiment, theheteroatom linker can be nitrogen, and the releasable linkers can beiminoalkylidenyl, carbonylalkylideniminyl, iminocycloalkylidenyl, andcarbonylcycloalkylideniminyl, wherein each of the releasable linkers isoptionally substituted with a substituent X², as defined below, and thereleasable linker is bonded to the nitrogen to form an hydrazone. In analternate configuration, the hydrazone may be acylated with a carboxylicacid derivative, an orthoformate derivative, or a carbamoyl derivativeto form various acylhydrazone releasable linkers.

Alternatively, the heteroatom linker can be oxygen, and the releasablelinkers can be alkylene(dialkylsilyl), alkylene(alkylarylsilyl),alkylene(diarylsilyl), (dialkylsilyl)aryl, (alkylarylsilyl)aryl, and(diarylsilyl)aryl, wherein each of the releasable linkers is optionallysubstituted with a substituent X², as defined below, and the releasablelinker is bonded to the oxygen to form a silanol.

In the above releasable linker embodiment, the drug can include anitrogen atom, the heteroatom linker can be nitrogen, and the releasablelinkers can be carbonylarylcarbonyl, carbonyl(carboxyaryl)carbonyl,carbonyl(biscarboxyaryl)carbonyl, and the releasable linker can bebonded to the heteroatom nitrogen to form an amide, and also bonded tothe drug nitrogen to form an amide.

In the above releasable linker embodiment, the drug can include anoxygen atom, the heteroatom linker can be nitrogen, and the releasablelinkers can be carbonylarylcarbonyl, carbonyl(carboxyaryl)carbonyl,carbonyl(biscarboxyaryl)carbonyl, and the releasable linker can bebonded to the heteroatom linker nitrogen to form an amide, and alsobonded to the drug oxygen to form an ester.

The substituents X² can be alkyl, alkoxy, alkoxyalkyl, hydroxy,hydroxyalkyl, amino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl,halo, haloalkyl, sulfhydrylalkyl, alkylthioalkyl, aryl, substitutedaryl, arylalkyl, substituted arylalkyl, heteroaryl, substitutedheteroaryl, carboxy, carboxyalkyl, alkyl carboxylate, alkyl alkanoate,guanidinoalkyl, R⁴-carbonyl, R⁵-carbonylalkyl, R⁶-acylamino, andR⁷-acylaminoalkyl, wherein R⁴ and R⁵ are each independently selectedfrom amino acids, amino acid derivatives, and peptides, and wherein R⁶and R⁷ are each independently selected from amino acids, amino acidderivatives, and peptides. In this embodiment the heteroatom linker canbe nitrogen, and the substituent X² and the heteroatom linker can betaken together with the releasable linker to which they are bound toform an heterocycle.

The heterocycles can be pyrrolidines, piperidines, oxazolidines,isoxazolidines, thiazolidines, isothiazolidines, pyrrolidinones,piperidinones, oxazolidinones, isoxazolidinones, thiazolidinones,isothiazolidinones, and succinimides.

The drug can include a nitrogen atom, and the releasable linker can behaloalkylenecarbonyl, optionally substituted with a substituent X², andthe releasable linker is bonded to the drug nitrogen to form an amide.

The drug can include an oxygen atom, and the releasable linker can behaloalkylenecarbonyl, optionally substituted with a substituent X², andthe releasable linker is bonded to the drug oxygen to form an ester.

The drug can include a double-bonded nitrogen atom, and in thisembodiment, the releasable linkers can be alkylenecarbonylamino and1-(alkylenecarbonylamino)succinimid-3-yl, and the releasable linker canbe bonded to the drug nitrogen to form an hydrazone.

The drug can include a sulfur atom, and in this embodiment, thereleasable linkers can be alkylenethio and carbonylalkylthio, and thereleasable linker can be bonded to the drug sulfur to form a disulfide.

The term “aryl” as used herein refers to an aromatic mono or polycyclicring of carbon atoms, such as phenyl, naphthyl, and the like.

The term “heteroaryl” as used herein refers to an aromatic mono orpolycyclic ring of carbon atoms and at least one heteroatom selectedfrom nitrogen, oxygen, and sulfur, such as pyridinyl, pyrimidinyl,indolyl, benzoxazolyl, and the like.

The term “substituted aryl” or “substituted heteroaryl” as used hereinrefers to aryl or heteroaryl substituted with one or more substituentsselected, such as halo, hydroxy, amino, alkyl or dialkylamino, alkoxy,alkylsulfonyl, cyano, nitro, and the like.

In addition, the following linkers are contemplated. It is understoodthat these linkers may be combined with each other and other space,heteroatom and releaseable linkers to prepare the conjugates describedherein. Illustrative linkers, and combinations of spacer and heteroatomlinkers include:

Illustrative linkers, and combinations of releasable and heteroatomlinkers include:

Illustrative folate receptor binding ligands include folic acid, folinicacid, pteropolyglutamic acid, and folate receptor-binding pteridinessuch as tetrahydropterins, dihydrofolates, tetrahydrofolates, and theirdeaza and dideaza analogs. The terms “deaza” and “dideaza” analogs referto the art-recognized analogs having a carbon atom substituted for oneor two nitrogen atoms in the naturally occurring folic acid structure,or analog or derivative thereof. For example, the deaza analogs includethe 1-deaza, 3-deaza, 5-deaza, 8-deaza, and 10-deaza analogs of folate.The dideaza analogs include, for example, 1,5-dideaza, 5,10-dideaza,8,10-dideaza, and 5,8-dideaza analogs of folate. Other folates useful ascomplex forming ligands for this invention are the folatereceptor-binding analogs aminopterin, amethopterin (methotrexate),N¹⁰-methylfolate, 2-deamino-hydroxyfolate, deaza analogs such as1-deazamethopterin or 3-deazamethopterin, and3′,5′-dichloro-4-amino-4-deoxy-N¹⁰-methylpteroylglutamic acid(dichloromethotrexate). The foregoing folic acid analogs and/orderivatives are conventionally termed “folate” or “folates,” reflectingtheir ability to bind with folate-receptors, and such ligands whenconjugated with exogenous molecules are effective to enhancetransmembrane transport, such as via folate-mediated endocytosis asdescribed herein. Other suitable ligands capable of binding to folatereceptors to initiate receptor-mediated endocytotic transport of thecomplex include anti-idiotypic antibodies to the folate receptor. Anexogenous molecule in complex with an anti-idiotypic antibody to afolate receptor is used to trigger transmembrane transport of thecomplex in accordance with the present invention.

Generally, any manner of forming a conjugate between the bivalent linker(L) and the folate receptor-binding ligand, or between the bivalentlinker (L) and the cell-growth inhibitor, antigen, or cytotoxin, oranalog or derivative thereof, including any intervening heteroatomlinkers, may be used. The conjugate may be formed by direct conjugationof any of these molecules, for example, through hydrogen, ionic, orcovalent bonds. Covalent bonding can occur, for example, through theformation of amide, ester, disulfide, or imino bonds between acid,aldehyde, hydroxy, amino, sulfhydryl, hydrazo, and like groups, such asthose described herein.

The spacer and/or releasable linker (i.e., cleavable linker) can be anybiocompatible linker. The cleavable linker can be, for example, a linkersusceptible to cleavage under the reducing or oxidizing conditionspresent in or on cells, a pH-sensitive linker that may be an acid-labileor base-labile linker, or a linker that is cleavable by biochemical ormetabolic processes, such as an enzyme-labile linker. Generally, thespacer and/or releasable linker comprises about 1 to about 50 atoms inlength, more typically about 2 to about 20 carbon atoms. It isappreciated that lower molecular weight linkers (i.e., those having anapproximate molecular weight of about 30 to about 300) may be employed.Precursors to such linkers are selected to have suitably reactive groupsat the points of attachment, such as nucleophilic or electrophilicfunctional groups, or both, optionally in a protected form with areadily cleavable protecting group to facilitate their use in synthesisof the intermediate species.

In another illustrative embodiment, the conjugate is a compound of thefollowing formula:

wherein:

R is —O—C═O.CR⁷R⁸R⁹;

R⁷ is hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms,alkynyl of 2-7 carbon atoms, —(CR¹²R¹³)_(f)OR¹⁰, —CF₃, —F, or —CO₂R¹⁰;

R¹⁰ is hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms,alkynyl of 2-7 carbon atoms, triphenylmethyl, benzyl, alkoxymethyl of2-7 carbon atoms, chloroethyl, or tetrahydropyranyl; R⁸ and R⁹ are takentogether to form X;

X is 2-phenyl-1,3,2-dioxaborinan-5-yl or2-phenyl-1,3,2-dioxaborinan-4-yl, wherein the phenyl may be optionallysubstituted;

R¹² and R¹³ are each, independently, hydrogen, alkyl of 1-6 carbonatoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms,trifluoromethyl, or —F;

f=0-6; and

L is as defined herein.

In another illustrative embodiment, the conjugate is a compound of thefollowing formula:

wherein R in each instance is the same or different and is independentlyselected from the group consisting of alkyl of 1-6 carbon atoms, phenyland benzyl; and L is as defined herein.

In another illustrative embodiment, the conjugate is a compound of thefollowing formula:

where L is as defined herein, and L is connected to the rapamycin oranalog or derivative thereof at either of (O*), and the other of (O*) issubstituted with R, wherein R is hydrogen or —(R^(a)—W—R^(b))_(n)—;

W is a linking group;

R^(a) is selected from the group consisting of carbonyl, —S(O)—,—S(O)₂—, —P(O)₂—, —P(O)(CH₃)—, —C(S)—, and —CH₂C(O)—;

R^(b) is a selected from the group consisting of carbonyl, —NH—, —S—,—CH₂—, and —O—; and

n=1-5.

In another illustrative embodiment, the conjugate is a compound of thefollowing formula:

where L is as defined herein, and L is connected to the rapamycin oranalog or derivative thereof at either of (O*), and the other of (O*) issubstituted with R, wherein R is hydrogen, thioalkyl of 1-6 carbonatoms, arylalkyl of 7-10 carbon atoms, hydroxyalkyl of 1-6 carbon atoms,dihydroxyalkyl of 1-6 carbon atoms, alkoxyalkyl of 2-12 carbon atoms,hydroxyalkoxyalkyl of 2-12 carbon atoms, acyloxyalkyl of 3-12 carbonatoms, aminoalkyl of 1-6 carbon atoms, alkylaminoalkyl of 1-6 carbonatoms per alkyl group, dialkylaminoalkyl of 1-6 carbon atoms per alkylgroup, alkoxycarbonylaminoalkyl of 3-12 carbon atoms, acylaminoalkyl of3-12 carbon atoms, alkenyl of 2-7 carbon atoms, arylsulfamidoalkylhaving 1-6 carbon atoms in the alkyl group, hydroxyalkylallyl of 4-9carbon atoms, dihydroxyalkylallyl of 4-9 carbon atoms, ordioxolanylallyl.

In another illustrative embodiment, the conjugate is a compound of thefollowing formula:

where L is as defined herein, and L is connected to the rapamycin oranalog or derivative thereof at either of (O*), and the other of (O*) issubstituted with R, wherein R is hydrogen or—CO(CR³R⁴)_(b)(CR⁵R⁶)_(d)CR⁷R⁸R⁹; where

R³ and R⁴ are each, independently, hydrogen, alkyl of 1-6 carbon atoms,alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms,trifluoromethyl, or F; R⁵ and R⁶ are each, independently, hydrogen,alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7carbon atoms, (CR³R⁴)_(f)OR¹⁰, CF₃, F, or CO₂R¹¹; R⁷ is hydrogen, alkylof 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbonatoms, (CR³R⁴)_(f)OR¹⁰, CF₃, F, or CO₂R¹¹;

R⁸ and R⁹ are each, independently, hydrogen, alkyl of 1-6 carbon atoms,alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms,(CR³R⁴)_(t)OR¹⁰, CF₃, F, or CO₂R¹¹;

R¹⁰ is hydrogen or COCH₂SCH₂CH₂(OCH₂CH₂)_(n)OCH₃;

R¹¹ is hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms,alkynyl of 2-7 carbon atoms, or phenylalkyl of 7-10 carbon atoms;

b=0-6; d=0-6; f=0-6; and n=5-450.

In another illustrative embodiment, the conjugate is a compound of thefollowing formula:

where L is as defined herein, and L is connected to the rapamycin oranalog or derivative thereof at either of (O*), and the other of (O*) issubstituted with R, wherein R is hydrogen or—CO(CR³R⁴)_(b)(CR⁵R⁶)_(d)CR⁷R⁸R⁹; where

R³ and R⁴ are each, independently, hydrogen, alkyl of 1-6 carbon atoms,alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms,trifluoromethyl, or F;

R⁵ and R⁶ are each, independently, hydrogen, alkyl of 1-6 carbon atoms,alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, (CR³R⁴)_(f)OH,CF₃, F, or CO₂R¹¹;

R⁷ is hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms,alkynyl of 2-7 carbon atoms, (CR³R⁴)_(f)OH, CF₃, F, or CO₂R¹¹;

R⁸ and R⁹ are each, independently, hydrogen, alkyl of 1-6 carbon atoms,alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, (CR³R⁴)_(f)OH,CF₃, F, or CO₂R¹¹;

R¹¹ is hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms,alkynyl of 2-7 carbon atoms, or phenylalkyl of 7-10 carbon atoms;

b=0-6; d=0-6; and f=0-6.

In one aspect of each of the foregoing, L includes an amino acid or apeptide. In another aspect of each of the foregoing, L includes aminoacids selected from cysteine, aspartic acid, glutamic acid, arginine,and lysine. It is to be understood that either enantiomer of such aminoacids may be included in such illustrative linkers in each instance. Inanother aspect of each of the foregoing, L includes a releasable linker.In one variation, the releasable linker comprises a disulfide bond. Inanother variation, the releasable linker comprises a carbonate.

In another illustrative embodiment, the conjugate is a compound of thefollowing formula:

where L is as defined herein, and L is connected to the rapamycin oranalog or derivative thereof at either of (O*). In one aspect, Lincludes an amino acid or a peptide. In another aspect, L includes aminoacids selected from cysteine, aspartic acid, glutamic acid, arginine,and lysine. It is to be understood that either enantiomer of such aminoacids may be included in such illustrative linkers in each instance. Inanother aspect, L includes a releasable linker. In one variation, thereleasable linker comprises a disulfide bond. In another variation, thereleasable linker comprises a carbonate.

In another illustrative embodiment, the conjugate is a compound of thefollowing formula:

where L is as defined herein, and L is connected to the rapamycin oranalog or derivative thereof at either of (O*). In one aspect, Lincludes an amino acid or a peptide. In another aspect, L includes aminoacids selected from cysteine, aspartic acid, glutamic acid, arginine,and lysine. It is to be understood that either enantiomer of such aminoacids may be included in such illustrative linkers in each instance. Inanother aspect, L includes a releasable linker. In one variation, thereleasable linker comprises a disulfide bond. In another variation, thereleasable linker comprises a carbonate.

In another illustrative embodiment, the conjugate is a compound of thefollowing formula:

where L is as defined herein. In one aspect, L includes an amino acid ora peptide. In another aspect, L includes amino acids selected fromcysteine, aspartic acid, glutamic acid, arginine, and lysine. It is tobe understood that either enantiomer of such amino acids may be includedin such illustrative linkers in each instance.

In another illustrative embodiment, the conjugate is a compound of thefollowing formula (EC0371; see also FIG. 3):

The compounds described herein may be prepared by general organicsynthetic reactions, such as those described in U.S. patent applicationSer. No. 10/765,336, the disclosure of which is incorporated herein byreference.

Briefly, the following chemical transformations are described forpreparing the compounds described herein.

General amide and ester formation. For example, where the heteroatomlinker is a nitrogen atom, and the terminal functional group present onthe spacer linker or the releasable linker is a carbonyl group, therequired amide group can be obtained by coupling reactions or acylationreactions of the corresponding carboxylic acid or derivative, where L isa suitably-selected leaving group such as halo, triflate,pentafluorophenoxy, trimethylsilyloxy, succinimide-N-oxy, and the like,and an amine, as illustrated in Scheme 1.

Coupling reagents include DCC, EDC, RRDQ, CGI, HBTU, TBTU, HOBT/DCC,HOBT/EDC, BOP-Cl, PyBOP, PyBroP, and the like. Alternatively, the parentacid can be converted into an activated carbonyl derivative, such as anacid chloride, a N-hydroxysuccinimidyl ester, a pentafluorophenyl ester,and the like. The amide-forming reaction can also be conducted in thepresence of a base, such as triethylamine, diisopropylethylamine,N,N-dimethyl-4-aminopyridine, and the like. Suitable solvents forforming amides described herein include CH₂Cl₂, CHCl₃, THF, DMF, DMSO,acetonitrile, EtOAc, and the like. Illustratively, the amides can beprepared at temperatures in the range from about −15° C. to about 80°C., or from about 0° C. to about 45° C. Amides can be formed from, forexample, nitrogen-containing aziridine rings, carbohydrates, andα-halogenated carboxylic acids. Illustrative carboxylic acid derivativesuseful for forming amides include compounds having the formulae:

and the like, where n is an integer such as 1, 2, 3, or 4.

Similarly, where the heteroatom linker is an oxygen atom and theterminal functional group present on the spacer linker or the releasablelinker is a carbonyl group, the required ester group can be obtained bycoupling reactions of the corresponding carboxylic acid or derivative,and an alcohol.

Coupling reagents include DCC, EDC, CDI, BOP, PyBOP, isopropenylchloroformate, EEDQ, DEAD, PPh₃, and the like. Solvents include CH₂Cl₂,CHCl₃, THF, DMF, DMSO, acetonitrile, EtOAc, and the like. Bases includetriethylamine, diisopropyl-ethylamine, and N,N-dimethyl-4-aminopyridine.Alternatively, the parent acid can be converted into an activatedcarbonyl derivative, such as an acid chloride, a N-hydroxysuccinimidylester, a pentafluorophenyl ester, and the like.

General ketal and acetal formation. Furthermore, where the heteroatomlinker is an oxygen atom, and the functional group present on the spacerlinker or the releasable linker is 1-alkoxyalkyl, the required acetal orketal group can be formed by ketal and acetal forming reactions of thecorresponding alcohol and an enol ether, as illustrated in Scheme 2.

Solvents include alcohols, CH₂Cl₂, CHCl₃, THF, diethylether, DMF, DMSO,acetonitrile, EtOAc, and the like. The formation of such acetals andketals can be accomplished with an acid catalyst. Where the heteroatomlinker comprises two oxygen atoms, and the releasable linker ismethylene, optionally substituted with a group X² as described herein,the required symmetrical acetal or ketal group can be illustrativelyformed by acetal and ketal forming reactions from the correspondingalcohols and an aldehyde or ketone, as illustrated in Scheme 3.

Alternatively, where the methylene is substituted with anoptionally-substituted aryl group, the required acetal or ketal may beprepared stepwise, where L is a suitably selected leaving group such ashalo, trifluoroacetoxy, triflate, and the like, as illustrated in Scheme4. The process illustrated in Scheme 4 is a conventional preparation,and generally follows the procedure reviewed by R. R. Schmidt et al.,Chem. Rev., 2000, 100, 4423-42, the disclosure of which is incorporatedherein by reference.

The resulting arylalkyl ether is treated with an oxidizing agent, suchas DDQ, and the like, to generate an intermediate oxonium ion that issubsequently treated with another alcohol to generate the acetal orketal.

General succinimide formation. Furthermore, where the heteroatom linkeris, for example, a nitrogen, oxygen, or sulfur atom, and the functionalgroup present on the spacer linker or the releasable linker is asuccinimide derivative, the resulting carbon-heteroatom bond can beformed by a Michael addition of the corresponding amine, alcohol, orthiol, and a maleimide derivative, where X is the heteroatom linker, asillustrated in Scheme 5.

Solvents for performing the Michael addition include THF, EtOAc, CH₂Cl₂,DMF, DMSO, H₂O and the like. The formation of such Michael adducts canbe accomplished with the addition of equimolar amounts of bases, such astriethylamine, Hünig's base or by adjusting the pH of water solutions to6.0-7.4. It is appreciated that when the heteroatom linker is an oxygenor nitrogen atom, reaction conditions may be adjusted to facilitate theMichael addition, such as, for example, by using higher reactiontemperatures, adding catalysts, using more polar solvents, such as DMF,DMSO, and the like, and activating the maleimide with silylatingreagents.

General silyloxy formation. Furthermore, where the heteroatom linker isan oxygen atom, and the functional group present on the spacer linker orthe releasable linker is a silyl derivative, the required silyloxy groupmay be formed by reacting the corresponding silyl derivative, and analcohol, where L is a suitably selected leaving group such as halo,trifluoroacetoxy, triflate, and the like, as illustrated in Scheme 6.

Silyl derivatives include properly functionalized silyl derivatives suchas vinylsulfonoalkyl diaryl, or diaryl, or alkyl aryl silyl chloride.Instead of a vinylsulfonoalkyl group, a β-chloroethylsulfonoalkylprecursor may be used. Any aprotic and anhydrous solvent and anynitrogen-containing base may serve as a reaction medium. The temperaturerange employed in this transformation may vary between −78° C. and 80°C.

General hydrazone formation. Furthermore, where the heteroatom linker isa nitrogen atom, and the functional group present on the spacer linkeror the releasable linker is an iminyl derivative, the required hydrazonegroup can be formed by reacting the corresponding aldehyde or ketone,and a hydrazine or acylhydrazine derivative, as illustrated in Scheme 7,equations (1) and (2) respectively.

Solvents that can be used include THF, EtOAc, CH₂Cl₂, CHCl₃, CCl₄, DMF,DMSO, MeOH and the like. The temperature range employed in thistransformation may vary between 0° C. and 80° C. Any acidic catalystsuch as a mineral acid, H₃CCOOH, F₃CCOOH, p-TsOH.H₂O, pyridiniump-toluene sulfonate, and the like can be used. In the case of theacylhydrazone in equation (2), the acylhydrazone may be prepared byinitially acylating hydrazine with a suitable carboxylic acid orderivative, as generally described above in Scheme 1, and subsequentlyreacting the acylhydrazide with the corresponding aldehyde or ketone toform the acylhydrazone. Alternatively, the hydrazone functionality maybe initially formed by reacting hydrazine with the correspondingaldehyde or ketone. The resulting hydrazone may subsequently be acylatedwith a suitable carboxylic acid or derivative, as generally describedabove in Scheme 1.

General disulfide formation. Furthermore, where the heteroatom linker isa sulfur atom, and the functional group present on the releasable linkeris an alkylenethiol derivative, the required disulfide group can beformed by reacting the corresponding alkyl or aryl sulfonylthioalkylderivative, or the corresponding heteroaryldithioalkyl derivative suchas a pyridin-2-yldithioalkyl derivative, and the like, with analkylenethiol derivative, as illustrated in Scheme 8.

Solvents that can be used are THF, EtOAc, CH₂Cl₂, CHCl₃, CCl₄, DMF,DMSO, and the like. The temperature range employed in thistransformation may vary between 0° C. and 80° C. The required alkyl oraryl sulfonylthioalkyl derivative may be prepared using art-recognizedprotocols, and also according to the method of Ranasinghe and Fuchs,Synth. Commun. 18(3), 227-32 (1988), the disclosure of which isincorporated herein by reference. Other methods of preparingunsymmetrical dialkyl disulfides are based on a transthiolation ofunsymmetrical heteroaryl-alkyl disulfides, such as 2-thiopyridinyl,3-nitro-2-thiopyridinyl, and like disulfides, with alkyl thiol, asdescribed in WO 88/01622, European Patent Application No. 0116208A1, andU.S. Pat. No. 4,691,024, the disclosures of which are incorporatedherein by reference.

General carbonate formation. Furthermore, where the heteroatom linker isan oxygen atom, and the functional group present on the spacer linker orthe releasable linker is an alkoxycarbonyl derivative, the requiredcarbonate group can be formed by reacting the correspondinghydroxy-substituted compound with an activated alkoxycarbonyl derivativewhere L is a suitable leaving group, as illustrated in Scheme 9.

Solvents that can be used are THF, EtOAc, CH₂Cl₂, CHCl₃, CCl₄, DMF,DMSO, and the like. The temperature range employed in thistransformation may vary between 0° C. and 80° C. Any basic catalyst suchas an inorganic base, an amine base, a polymer bound base, and the likecan be used to facilitate the reaction.

General semicarbazone formation. Furthermore, where the heteroatomlinker is a nitrogen atom, and the functional group present on onespacer linker or the releasable linker is an iminyl derivative, and thefunctional group present on the other spacer linker or the otherreleasable linker is an alkylamino or arylaminocarbonyl derivative, therequired semicarbazone group can be formed by reacting the correspondingaldehyde or ketone, and a semicarbazide derivative, as illustrated inScheme 10.

Solvents that can be used are THF, EtOAc, CH₂Cl₂, CHCl₃, CCl₄, DMF,DMSO, MeOH and the like. The temperature range employed in thistransformation may vary between 0° C. and 80° C. Any acidic catalystsuch as a mineral acid, H₃CCOOH, F₃CCOOH, p-TsOH.H₂O, pyridiniump-toluene sulfonate, and the like can be used. In addition, in formingthe semicarbazone, the hydrazone functionality may be initially formedby reacting hydrazine with the corresponding aldehyde or ketone. Theresulting hydrazone may subsequently by acylated with an isocyanate or acarbamoyl derivative, such as a carbamoyl halide, to form thesemicarbazone. Alternatively, the corresponding semicarbazide may beformed by reacting hydrazine with an isocyanate or carbamoyl derivative,such as a carbamoyl halide to form a semicarbazide. Subsequently, thesemicarbazide may be reacted with the corresponding aldehyde or ketoneto form the semicarbazone.

General sulfonate formation. Furthermore, where the heteroatom linker isan oxygen atom, and the functional group present on the spacer linker orthe releasable linker is sulfonyl derivative, the required sulfonategroup can be formed by reacting the corresponding hydroxy-substitutedcompound with an activated sulfonyl derivative where L is a suitableleaving group such as halo, and the like, as illustrated in Scheme 11.

Solvents that can be used are THF, EtOAc, CH₂Cl₂, CHCl₃, CCl₄, and thelike. The temperature range employed in this transformation may varybetween 0° C. and 80° C. Any basic catalyst such as an inorganic base,an amine base, a polymer bound base, and the like can be used tofacilitate the reaction.

General formation of folate-peptides. The folate-containing peptidylfragment Pte-Glu-(AA)_(n)-NH(CHR₂)CO₂H (3) is prepared by apolymer-supported sequential approach using standard methods, such asthe Fmoc-strategy on an acid-sensitive Fmoc-AA-Wang resin (1), as shownin Scheme 12.

In this illustrative embodiment of the processes described herein, R₁ isFmoc, R₂ is the desired appropriately-protected amino acid side chain,and DIPEA is diisopropylethylamine. Standard coupling procedures, suchas PyBOP and others described herein or known in the art are used, wherethe coupling agent is illustratively applied as the activating reagentto ensure efficient coupling. Fmoc protecting groups are removed aftereach coupling step under standard conditions, such as upon treatmentwith piperidine, tetrabutylammonium fluoride (TBAF), and the like.Appropriately protected amino acid building blocks, such asFmoc-Glu-OtBu, N¹⁰-TFA-Pte-OH, and the like, are used, as described inScheme 12, and represented in step (b) by Fmoc-AA-OH. Thus, AA refers toany amino acid starting material that is appropriately protected. It isto be understood that the term amino acid as used herein is intended torefer to any reagent having both an amine and a carboxylic acidfunctional group separated by one or more carbons, and includes thenaturally occurring alpha and beta amino acids, as well as amino acidderivatives and analogs of these amino acids. In particular, amino acidshaving side chains that are protected, such as protected serine,threonine, cysteine, aspartate, and the like may also be used in thefolate-peptide synthesis described herein. Further, gamma, delta, orlonger homologous amino acids may also be included as starting materialsin the folate-peptide synthesis described herein. Further, amino acidanalogs having homologous side chains, or alternate branchingstructures, such as norleucine, isovaline, β-methyl threonine, β-methylcysteine, β,β-dimethyl cysteine, and the like, may also be included asstarting materials in the folate-peptide synthesis described herein.

The coupling sequence (steps (a) & (b)) involving Fmoc-AA-OH isperformed “n” times to prepare solid-support peptide 2, where n is aninteger and may equal 0 to about 100. Following the last coupling step,the remaining Fmoc group is removed (step (a)), and the peptide issequentially coupled to a glutamate derivative (step (c)), deprotected,and coupled to TFA-protected pteroic acid (step (d)). Subsequently, thepeptide is cleaved from the polymeric support upon treatment withtrifluoroacetic acid, ethanedithiol, and triisopropylsilane (step (e)).These reaction conditions result in the simultaneous removal of thet-Bu, t-Boc, and Trt protecting groups that may form part of theappropriately-protected amino acid side chain. The TFA protecting groupis removed upon treatment with base (step (O) to provide thefolate-containing peptidyl fragment 3.

In another method of treatment embodiment, the group D in the targetedconjugate V-L-D, comprises an antigen (i.e., a compound that isadministered for the purpose of eliciting an immune response in vivo),the ligand-antigen conjugates being effective to “label” the populationof proximal tubule cells responsible for disease pathogenesis in thepatient suffering from the kidney disease for specific elimination by anendogenous immune response or by co-administered antibodies. The use ofligand-antigen conjugates in the method of treatment described hereinworks to enhance an immune response-mediated elimination of the proximaltubule cells proliferating abnormally that overexpress the ligandreceptor. Such elimination can be effected through an endogenous immuneresponse or by a passive immune response effected by co-administeredantibodies.

The methods of treatment involving the use of ligand-antigen conjugatesare described in U.S. patent application Ser. Nos. 09/822,379,10/138,275, and PCT Application Serial No. PCT/US04/014097, eachincorporated herein by reference.

The endogenous immune response can include a humoral response, acell-mediated immune response, and any other immune response endogenousto the host animal, including complement-mediated cell lysis,antibody-dependent cell-mediated cytotoxicity (ADCC), antibodyopsonization leading to phagocytosis, clustering of receptors uponantibody binding resulting in signaling of apoptosis, antiproliferation,or differentiation, and direct immune cell recognition of the deliveredantigen (e.g., a hapten). It is also contemplated that the endogenousimmune response may employ the secretion of cytokines that regulate suchprocesses as the multiplication, differentiation, and migration ofimmune cells. The endogenous immune response may include theparticipation of such immune cell types as B cells, T cells, includinghelper and cytotoxic T cells, macrophages, natural killer cells,neutrophils, LAK cells, and the like.

The humoral response can be a response induced by such processes asnormally scheduled vaccination, or active immunization with a naturalantigen or an unnatural antigen or hapten, e.g., fluoresceinisothiocyanate (FITC) or dinitrophenyl (DNP), with the unnatural antigeninducing a novel immunity. Active immunization involves multipleinjections of the unnatural antigen or hapten scheduled outside of anormal vaccination regimen to induce the novel immunity. The humoralresponse may also result from an innate immunity where the host animalhas a natural preexisting immunity, such as an immunity to α-galactosylgroups.

Alternatively, a passive immunity may be established by administeringantibodies to the host animal such as natural antibodies collected fromserum or monoclonal antibodies that may or may not be geneticallyengineered antibodies, including humanized antibodies. The utilizationof a particular amount of an antibody reagent to develop a passiveimmunity, and the use of a ligand-antigen conjugate wherein thepassively administered antibodies are directed to the antigen, wouldprovide the advantage of a standard set of reagents to be used in caseswhere a patient's preexisting antibody titer to potential antigens isnot therapeutically useful. The passively administered antibodies may be“co-administered” with the ligand-antigen conjugate, andco-administration is defined as administration of antibodies at a timeprior to, at the same time as, or at a time following administration ofthe ligand-antigen conjugate.

The preexisting antibodies, induced antibodies, or passivelyadministered antibodies will be redirected to the proximal tubule cellsproliferating abnormally by binding of the ligand-antigen conjugates tothe proximal tubule cell populations overexpressing the receptor for theligand, and such pathogenic cells are killed or eliminated or reduced innumber by complement-mediated lysis, ADCC, antibody-dependentphagocytosis, or antibody clustering of receptors. The cytotoxic processmay also involve other types of immune responses, such as cell-mediatedimmunity.

Acceptable antigens for use in preparing the conjugates used in themethod of treatment described herein are antigens that are capable ofeliciting antibody production in a patient or animal or that havepreviously elicited antibody production in a patient or animal,resulting in a preexisting immunity, or that constitute part of theinnate immune system. Alternatively, antibodies directed against theantigen may be administered to the patient or animal to establish apassive immunity. Suitable antigens for use in the invention includeantigens or antigenic peptides against which a preexisting immunity hasdeveloped via normally scheduled vaccinations or prior natural exposureto such agents such as polio virus, tetanus, typhus, rubella, measles,mumps, pertussis, tuberculosis and influenza antigens, and α-galactosylgroups. In such cases, the ligand-antigen conjugates will be used toredirect a previously acquired humoral or cellular immunity to apopulation of proximal tubule cells proliferating abnormally in thepatient or animal for elimination of the proximal tubule cells orreduction in number or inactivation, completely or partially.

Other suitable immunogens include antigens or antigenic peptides towhich the host animal has developed a novel immunity throughimmunization against an unnatural antigen or hapten, for example,fluorescein isothiocyanate (FITC) or dinitrophenyl, and antigens againstwhich an innate immunity exists, for example, super antigens and muramyldipeptide.

The proximal tubule cell-binding ligands and antigens, cytotoxic agents,and cell growth inhibitors, or diagnostic markers, as the case may be,in forming conjugates for use in accordance with the methods describedherein can be conjugated by using any art-recognized method for forminga complex. This can include covalent, ionic, or hydrogen bonding of theligand V to the group D compound, either directly or indirectly via alinking group such as a divalent linker. The conjugate is typicallyformed by covalent bonding of the ligand to the targeted entity throughthe formation of amide, ester or imino bonds between acid, aldehyde,hydroxy, amino, or hydrazo groups on the respective components of thecomplex or, for example, by the formation of disulfide bonds. Methods oflinking binding ligands to antigens, cytotoxic agents, or cell growthinhibitors, or diagnostic markers are described in U.S. patentapplication Ser. Nos. 10/765,336 and 60/590,580, each incorporatedherein by reference.

Alternatively, as mentioned above, the ligand complex can be onecomprising a liposome wherein the targeted entity (that is, thediagnostic marker, or the antigen, cytotoxic agent or cell growthinhibitor) is contained within a liposome which is itself covalentlylinked to the binding ligand. Other nanoparticles, dendrimers,derivatizable polymers or copolymers that can be linked to therapeuticor diagnostic markers useful in the treatment and diagnosis of kidneydisease states can also be used in targeted conjugates.

In one embodiment of the invention the ligand is folic acid, an analogof folic acid, or any other folate receptor binding molecule, and thefolate ligand is conjugated to the targeted entity by a procedure thatutilizes trifluoroacetic anhydride to prepare γ-esters of folic acid viaa pteroyl azide intermediate. This procedure results in the synthesis ofa folate ligand, conjugated to the targeted entity only through theγ-carboxy group of the glutamic acid groups of folate. Alternatively,folic acid analogs can be coupled through the α-carboxy moiety of theglutamic acid group or both the α and γ carboxylic acid entities.

The therapeutic methods described herein can be used to slow theprogress of disease completely or partially. Alternatively, thetherapeutic methods described herein can eliminate or preventreoccurrence of the disease state.

The conjugates used in accordance with the methods described herein ofthe formula V-L-D are used in one aspect to formulate therapeutic ordiagnostic compositions, for administration to a patient or animal,wherein the compositions comprise effective amounts of the conjugate andan acceptable carrier therefor. Typically such compositions areformulated for parenteral use. The amount of the conjugate effective foruse in accordance with the methods described herein depends on manyparameters, including the nature of the disease being treated ordiagnosed, the molecular weight of the conjugate, its route ofadministration and its tissue distribution, and the possibility ofco-usage of other therapeutic or diagnostic agents. The effective amountto be administered to a patient or animal is typically based on bodysurface area, patient weight and physician assessment of patientcondition. An effective amount can range from about to 1 ng/kg to about1 mg/kg, more typically from about 1 μg/kg to about 500 μg/kg, and mosttypically from about 1 μg/kg to about 100 μg/kg.

Any effective regimen for administering the ligand conjugates can beused. For example, the ligand conjugates can be administered as singledoses, or they can be divided and administered as a multiple-dose dailyregimen. Further, a staggered regimen, for example, one to three daysper week can be used as an alternative to daily treatment, and such anintermittent or staggered daily regimen is considered to be equivalentto every day treatment and within the scope of this disclosure. In oneembodiment, the patient or animal is treated with multiple injections ofthe ligand conjugate wherein the targeted entity is an antigen or acytotoxic agent or a cell growth inhibitor to eliminate the populationof pathogenic proximal tubule cells. In one embodiment, the patient oranimal is treated, for example, injected multiple times with the ligandconjugate at, for example, 12-72 hour intervals or at 48-72 hourintervals. Additional injections of the ligand conjugate can beadministered to the patient or animal at intervals of days or monthsafter the initial injections, and the additional injections preventrecurrence of disease. Alternatively, the ligand conjugates may beadministered prophylactically to prevent the occurrence of disease inpatients or animals known to be disposed to development of kidneydisease states. In one embodiment, more than one type of ligandconjugate can be used, for example, the patient or animal may bepre-immunized with fluorescein isothiocyanate and dinitrophenyl andsubsequently treated with fluorescein isothiocyanate and dinitrophenyllinked to the same or different targeting ligands in a co-dosingprotocol.

The ligand conjugates are administered in one aspect parenterally andmost typically by intraperitoneal injections, subcutaneous injections,intramuscular injections, intravenous injections, intradermalinjections, or intrathecal injections. The ligand conjugates can also bedelivered to a patient or animal using an osmotic pump. Examples ofparenteral dosage forms include aqueous solutions of the conjugate, forexample, a solution in isotonic saline, 5% glucose or other well-knownpharmaceutically acceptable liquid carriers such as alcohols, glycols,esters and amides. The parenteral compositions for use in accordancewith this invention can be in the form of a reconstitutable lyophilizatecomprising the one or more doses of the ligand conjugate. In anotheraspect, the ligand conjugates can be formulated as one of any of anumber of prolonged release dosage forms known in the art such as, forexample, the biodegradable carbohydrate matrices described in U.S. Pat.Nos. 4,713,249; 5,266,333; and 5,417,982, the disclosures of which areincorporated herein by reference. The ligand conjugates can also beadministered topically such as in an ointment or a lotion, for example,or in a patch form.

The following examples are illustrative embodiments only and are notintended to be limiting.

Example 1 Materials

Fmoc-protected amino acid derivatives, trityl-protected cysteine2-chlorotrityl resin (H-Cys(Trt)-2-ClTrt resin #04-12-2811),Fmoc-lysine(4-methyltrityl) wang resin,2-(1H-benzotriaxol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphage(HBTU) and N-hydroxybenzotriazole can be purchased from Novabiochem (LaJolla, Calif.). N¹⁰-trifluoroacetylpteroic acid can be purchased fromSigma, St. Louis, Mo.

Example 2 Synthesis of Folate-Cysteine

Standard Fmoc peptide chemistry can be used to synthesizefolate-cysteine with the cysteine attached to the γ-COOH of folic acid.The sequence Cys-Glu-Pteroic acid (Folate-Cys) will be constructed byFmoc chemistry with HBTU and N-hydroxybenzotriazole as the activatingagents along with diisopropyethylamine as the base and 20% piperidine indimethylformamide (DMF) for deprotection of the Fmoc groups. Anα-t-Boc-protected N-α-Fmoc-L-glutamic acid will be linked to atrityl-protected Cys linked to a 2-Chlorotrityl resin.N¹⁰-trifluoroacetylpteroic acid was then attached to the γ-COOH of Glu.The Folate-Cys was cleaved from the resin using a 92.5% trifluoroaceticacid-2.5% water-2.5% triisopropylsilane-2.5% ethanedithio solution.Diethyl ether will be used to precipitate the product, and theprecipitant was collected by centrifugation. The product will be washedtwice with diethyl ether and dried under vacuum overnight. To remove theN¹⁰-trifluoracetyl protecting group, the product will be dissolved in a10% ammonium hydroxide solution and stirred for 30 min at roomtemperature. The solution will be kept under a stream of nitrogen theentire time in order to prevent the cysteine from forming disulfides.After 30 minutes, hydrochloric acid will be added to the solution untilthe compound precipitates. The product will be collected bycentrifugation and lyophilized. The product will be analyzed andconfirmed by mass spectroscopic analysis.

Example 3 Synthesis of Folate-R-Phycoerythrin

Folate-phycoerythrin will be synthesized by following a procedurepublished by Kennedy M. D. et al. in Pharmaceutical Research, Vol.20(5); 2003. Briefly, a 10-fold excess of folate-cysteine will be addedto a solution of R-phycoerythrin pyridyldisulfide (Sigma, St. Louis,Mo.) in phosphate buffered saline (PBS), pH 7.4. The solution will beallowed to react overnight at 4° C. and the labeled protein (Mr ˜260kDa) will be purified by gel filtration chromatography using a G-15desalting column. The folate labeling will be confirmed by fluorescencemicroscopy of M109 cells incubated with folate-phycoerythrin in thepresence and absence of 100-fold excess of folic acid. After a 1-hincubation and 3 cells washes with PBS, the treated cells will beintensely fluorescent, while the sample in the presence of excess folicacid will show little cellular fluorescence.

Example 4 Synthesis of Folate-Fluorescein

Folate-FITC will be synthesized as described by Kennedy, M. D. et al. inPharmaceutical Research, Vol. 20(5); 2003.

Example 5 Liposome Preparation

Liposomes will be prepared following methods by Leamon et al. inBioconjugate Chemistry 2003, 14, 738-747. Briefly, lipids andcholesterol will be purchased from Avanti Polar Lipids (Alabaster,Ala.). Folate-targeted liposomes will consist of 40 mole % cholesterol,either 4 mole % or 6 mole % polyethyleneglycol (Mr˜2000)-derivatizedphosphatidylethanolamine (PEG2000-PE, Nektar AL, Huntsville, Ala.),either 0.03 mole % or 0.1 mole % folate-cysteine-PEG3400-PE and theremaining mole % will be composed of egg phosphatidylcholine.Non-targeted liposomes will be prepared identically with the absence offolate-cysteine-PEG3400-PE. Lipids in chloroform will be dried to a thinfilm by rotary evaporation and then rehydrated in PBS containing thedrug. Rehydration will be accomplished by vigorous vortexing followed by10 cycles of freezing and thawing. Liposomes will be extruded 10 timesthrough a 50 nm pore size polycarbonate membrane using a high-pressureextruder (Lipex Biomembranes, Vancouver, Canada).

Example 6 Synthesis of Folate-Saporin

The protein saporin will be purchased from Sigma (St. Louis, Mo.).Folate-saporin will be prepared following folate-protein conjugationmethods published by Leamon and Low in The Journal of BiologicalChemistry 1992, 267(35); 24966-24971. Briefly, folic acid will bedissolved in DMSO and incubated with a 5 fold molar excess of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide for 30 minutes at roomtemperature. The saporin will be dissolved in 100 mM KH₂PO₄, 100 mMboric acid, pH 8.5. A 10-fold molar excess of the “activated” vitaminwill be added to the protein solution and the labeling reaction wasallowed to proceed for 4 hours. Unreacted material will be separatedfrom the labeled protein using a Sephadex G-25 column equilibrated inphosphate-buffered saline, pH 7.4.

Example 7 Synthesis of Folate-Peptides

Generally, the reagents shown in the following table were used in thepreparation of this example and other examples:

Reagent (mmol) equivalents Amount H-Cys(4-methoxytrityl)-2- 0.56 1 1.0 gchlorotrityl-Resin (loading 0.56 mmol/g) Fmoc-β-aminoalanine(NH- 1.12 20.653 g MTT)-OH Fmoc-Asp(OtBu)-OH 1.12 2 0.461 g Fmoc-Asp(OtBu)-OH 1.122 0.461 g Fmoc-Asp(OtBu)-OH 1.12 2 0.461 g Fmoc-Glu-OtBu 1.12 2 0.477 gN¹⁰TFA-Pteroic Acid 0.70 1.25 0.286 g (dissolve in 10 ml DMSO) DIPEA2.24 4 0.390 mL PyBOP 1.12 2 0.583 g

The coupling step was performed as follows: In a peptide synthesisvessel add the resin, add the amino acid solution, DIPEA, and PyBOP.Bubble argon for 1 hr. and wash 3× with DMF and IPA. Use 20% piperidinein DMF for Fmoc deprotection, 3× (10 min), before each amino acidcoupling. Continue to complete all 6 coupling steps. At the end wash theresin with 2% hydrazine in DMF 3× (5 min) to cleave TFA protecting groupon Pteroic acid.

Cleave the peptide analog from the resin using the following reagent,92.5% (50 ml) TFA, 2.5% (1.34 ml) H₂O, 2.5% (1.34 ml)Triisopropylsilane, 2.5% (1.34 ml) ethanedithiol, the cleavage step wasperformed as follows: Add 25 ml cleavage reagent and bubble for 1.5 hr,drain, and wash 3× with remaining reagent. Evaporate to about 5 mL andprecipitate in ethyl ether. Centrifuge and dry. Purification wasperformed as follows: Column—Waters NovaPak C₁₈ 300×19 mm; Buffer A=10mM Ammonium Acetate, pH 5; B=CAN; 1% B to 20% B in 40 minutes at 15ml/min, to 350 mg (64%); HPLC-RT 10.307 min., 100% pure, ¹H HMR spectrumconsistent with the assigned structure, and MS (ES−): 1624.8, 1463.2,1462.3, 977.1, 976.2, 975.1, 974.1, 486.8, 477.8.

Example 8 Synthesis of Folate-γ-Asp-Arg-Asp-Asp-Cys

According to the general procedure of the prior example and Scheme 12,Wang resin bound 4-methoxytrityl (MTT)-protected Cys-NH₂ was reactedaccording to the following sequence: 1) a. Fmoc-Asp(OtBu)-OH, PyBOP,DIPEA; b. 20% Piperidine/DMF; 2) a. Fmoc-Asp(OtBu)-OH, PyBOP, DIPEA; b.20% Piperidine/DMF; 3) a. Fmoc-Arg(Pbf)-OH, PyBOP, DIPEA; b. 20%Piperidine/DMF; 4) a. Fmoc-Asp(OtBu)-OH, PyBOP, DIPEA; b. 20%Piperidine/DMF; 5) a. Fmoc-Glu-OtBu, PyBOP, DIPEA; b. 20%Piperidine/DMF; 6) N¹⁰-TFA-pteroic acid, PyBOP, DIPEA. The MTT, tBu, andPbf protecting groups were removed with TFA/H₂O/TIPS/EDT(92.5:2.5:2.5:2.5), and the TFA protecting group was removed withaqueous NH₄OH at pH=9.3. Selected ¹H NMR (D₂O) δ (ppm) 8.68 (s, 1H, FAH-7), 7.57 (d, 2H, J=8.4 Hz, FA H-12 &16), 6.67 (d, 2H, J=9 Hz, FA H-13& 15), 4.40-4.75 (m, 5H), 4.35 (m, 2H), 4.16 (m, 1H), 3.02 (m, 2H),2.55-2.95 (m, 8H), 2.42 (m, 2H), 2.00-2.30 (m, 2H), 1.55-1.90 (m, 2H),1.48 (m, 2H); MS (ESI, m+H⁺) 1046.

Example 9 Synthesis of Folate-γ-Asp-Asp-Asp-(β-NH₂-Ala)-Cys

According to the general procedure of the prior example and Scheme 12,Wang resin bound 4-methoxytrityl (MTT)-protected Cys-NH₂ was reactedaccording to the following sequence: 1) a.Fmoc-β-aminoalanine(NH-MTT)-OH, PyBOP, DIPEA; b. 20% Piperidine/DMF; 2)a. Fmoc-Asp(OtBu)-OH, PyBOP, DIPEA; b. 20% Piperidine/DMF; 3) a.Fmoc-Asp(OtBu)-OH, PyBOP, DIPEA; b. 20% Piperidine/DMF; 4) a.Fmoc-Asp(OtBu)-OH, PyBOP, DIPEA; b. 20% Piperidine/DMF; 5) a.Fmoc-Glu-OtBu, PyBOP, DIPEA; b. 20% Piperidine/DMF; 6) N¹⁰-TFA-pteroicacid, PyBOP, DIPEA. The MTT, tBu, and TFA protecting groups were removedwith a. 2% hydrazine/DMF; b. TFA/H₂O/TIPS/EDT (92.5:2.5:2.5:2.5).

Example 10 Synthesis of Folate-α-Asp-Arg-Asp-Asp-Cys

According to the general procedure of the prior example and Scheme 12,Wang resin bound MTT-protected Cys-NH₂ was reacted according to thefollowing sequence: 1) a. Fmoc-Asp(OtBu)-OH, PyBOP, DIPEA; b. 20%Piperidine/DMF; 2) a. Fmoc-Asp(OtBu)-OH, PyBOP, DIPEA; b. 20%Piperidine/DMF; 3) a. Fmoc-Arg(Pbf)-OH, PyBOP, DIPEA; b. 20%Piperidine/DMF; 4) a. Fmoc-Asp(OtBu)-OH, PyBOP, DIPEA; b. 20%Piperidine/DMF; 5) a. Fmoc-Glu(γ-OtBu)-OH, PyBOP, DIPEA; b. 20%Piperidine/DMF; 6) N¹⁰-TFA-pteroic acid, PyBOP, DIPEA. The MTT, tBu, andPbf protecting groups were removed with TFA/H₂O/TIPS/EDT(92.5:2.5:2.5:2.5), and the TFA protecting group was removed withaqueous NH₄OH at pH=9.3. The ¹H NMR spectrum was consistent with theassigned structure.

Example 11 Synthesis of Folate-γ-D-Asp-D-Arg-D-Asp-D-Asp-D-Cys

According to the general procedure of the prior example and Scheme 12,Wang resin bound MTT-protected D-Cys-NH₂ was reacted according to thefollowing sequence: 1) a. Fmoc-D-Asp(OtBu)-OH, PyBOP, DIPEA; b. 20%Piperidine/DMF; 2) a. Fmoc-D-Asp(OtBu)-OH, PyBOP, DIPEA; b. 20%Piperidine/DMF; 3) a. Fmoc-D-Arg(Pbf)-OH, PyBOP, DIPEA; b. 20%Piperidine/DMF; 4) a. Fmoc-D-Asp(OtBu)-OH, PyBOP, DIPEA; b. 20%Piperidine/DMF; 5) a. Fmoc-D-Glu-OtBu, PyBOP, DIPEA; b. 20%Piperidine/DMF; 6) N¹⁰-TFA-pteroic acid, PyBOP, DIPEA. The MTT, tBu, andPbf protecting groups were removed with TFA/H₂O/TIPS/EDT(92.5:2.5:2.5:2.5), and the TFA protecting group was removed withaqueous NH₄OH at pH=9.3. The ¹H NMR spectrum was consistent with theassigned structure.

Example 12 Synthesis of Folate-Rapamycin (EC0371)

This example was prepared according to the prior scheme and theprocesses described herein.

Example 13 Animal Models

To test the ligand-cytotoxin, ligand-antigen, and ligand-cell growthinhibitor conjugates in animal models of PKD, well-established animalmodels will be used. Those animal models are described in Shillingford,et al., PNAS 103: 5466-5471 (2006), Piontek, et al., J. Am. Soc.Nephrol. vol. 15: 3035-3043 (2004), Brown, et al., Kidney Int. vol. 63:1220-1229 (2003), and Nauta, et al., Pediatr. Nephrol. vol. 7: 163-172(1993), incorporated herein by reference.

Example 14 Immunofluorescence

The ability of folate-conjugated rapamycin to inhibit the mTOR pathwaywas tested in KB cells using immunostaining for P-S6 as a marker (seeFIG. 6). The immunostaining procedure was performed according to thefollowing protocol:

1. Aspirate media from cells and immediately add 1 ml/well of 10%neutral-buffered formalin (NBF) to each well.

2. Fix cells for 15 minutes at room temp with gentle orbital shaking.

3. Aspirate NBF from cells and wash briefly with 2 changes (1 ml/well)of 1×PBS.

4. Aspirate PBS and add 1 ml of quench solution and quench for 10minutes at room temperature with gentle orbital shaking.

5. Aspirate quench and wash briefly with 2 changes (1 ml/well) of 1×PBS.

6. Aspirate PBS and add 1 ml/well cell block/permeabilization (CBP)solution and incubate for 30 minutes at 37° C.

7. Prepare P-S6 (S235/6)/(3-tubulin antibody solution by dilutingantibody 1:200 in CBP solution (for 12 coverslips make 1194 ul CBP+6 ulP-S6 and β-tubulin). Mix thoroughly.

8. Remove the lid from the cell culture dish and place in a humidifiedchamber. Cut parafilm to the size of the lid and press firmly on to thelid.

9. Using a pair of needle-nose tweezers transfer a coverslip, cell-sideup, to its corresponding well position on the lid. Pipet 150 μl ofP-S6/β-tubulin antibody solution onto the coverslip. Repeat for allremaining coverslips.

10. Close the lid and incubate overnight at 4° C.

11. The following day, make 100 ml cell wash (CW) solution.

12. Pipet 1 ml CW solution into each well of a fresh 12-well plate.Using two pairs of tweezers, one placed on the back of the coverslip,carefully pick up the coverslip and place back in the correspondingwell.

13. Incubate for 5 minutes with gentle orbital shaking. Aspirate andrepeat wash 2×.

14. During washes, dilute fluorescent-conjugated anti-rabbit FITC andanti-mouse TXR secondary antibodies 1:200 in CBP. Centrifuge 10 minutesat 4° C., 13,000 rpm, to remove aggregates.

15. After washing, repeat steps 8 and 9 with dilutedfluorescent-conjugated secondary antibody solution. Incubate for 1 hourat 37° C.

16. Repeat steps 12 and 13.

17. Rinse 1× with 1×PBS.

18. Aspirate and wash 2×3 minutes with 1×PBS+0.1% Triton-X 100 withgentle orbital shaking.

19. Aspirate and rinse 2× with 1×PBS.

20. Aspirate and add 1 ml of 10% NBF to post-fix secondary antibodies.Incubate for 10 minutes at room temperature with gentle orbital shaking.

21. Aspirate and wash 1×5 minutes with 1×PBS.

22. Aspirate and add 1 ml of 1×PBS+DAPI (1 mg/ml stock, 1:50,000dilution). Incubate for 5-10 minutes.

23. Thaw Prolong Gold mounting medium. Dispense two drops on a slide.Using needle-nose tweezers remove individual coverslips, wipe excesssolution from backside and place on top of mounting medium, cell-sidedown. Gently squeeze out any air bubbles with the opposite end of thetweezers.

25. Allow mounting medium to harden for at least 1 hour, preferablyovernight. View slides under a suitable microscope equipped forfluorescence. Store slides at −20° C.

Example 15 Folate Receptor Immunohistochemistry

Immunohistochemistry was performed as described in PCT Publ. No.WO/2006/105141, incorporated herein by reference. As shown in FIGS. 1and 2, monoclonal and polyclonal antibodies to the folate receptor staincysts in polycystic kidney disease tissues indicating folate receptoroverexpression in the cells that form PKD cysts (see also Table 1below).

TABLE 1 Polycystic Kidney Tissue from Mice and Humans Polycystic KidneyDisease IHC Results Specimen ID 3+ 2+ 1+ 0 PKD PKD Case 7 10%  30% 40%20% PKD Case 8 0% 30% 60% 10% PKD Case 9 0% 10% 20% 70% PKD Case 10 0% 0% 40% 60% Control N1 10%  20% 30% 40% Kidney N3 Tissue was not normalkidney N4 30%  30% 30% 10% Serous ITOC02407A 0% 20% 50% 30% OVCAITOC02463A 10%  20% 10% 60% ITOC02556A 0%  0%  0% 100%  PKD ORPK666B10%  10% 50% 30% BPK6468D 0% 10% 20% 70% BPK6467B 0% 20% 40% 40% ControlORPK665B 0%  0% 40% 60% Kidney ORPK667B 0%  5% 80% 15% BPK6466B 0%  5%75% 20% BPK6469B 0%  0%  0% 100% 

Example 16 Relative Affinity Assay

Binding assays were run to determine the relative affinities of EC0371and folic acid at the folate receptor. KB cells were incubated for 1hour at 37° C. with 100 nM ³H-folic acid in the presence and absence ofincreasing competitor concentrations. As shown in FIG. 4 (error barsrepresent 1 standard deviation (n=3)), the relative affinity of EC0371at the folate receptor is 0.5 compared to a relative affinity of 1.0 forfolic acid.

Example 17 Cell Viability

Cell viability was examined in KB cells following incubation for 16hours in Rapamycin (2, 10, and 50 nM), EC0371 (2, 10, and 50 nM), DMSO(diluent), and media alone (FIG. 5). At 24 hours, neither rapamycin norEC0371 was found to be cytotoxic at any of the concentrations tested.

Example 18 P-S6 and P-S6K Immunoblots

Folate-rapamycin was found to be highly effective in inhibiting mTOR incultured cells. Folate receptor-positive KB cells were treated witheither unconjugated rapamycin (2, 10, or 50 nM) or folate-rapamycin (2,10, or 50 nM) for 16 hours. The activity of mTOR was determined byimmunoblotting using phospho-specific antibodies against P-S6 and P-S6K(FIG. 7).

Example 19 Therapeutic Effects of EC0371 In Vivo

The therapeutic effect of EC0371 (folate-conjugated rapamycin) wastested on in vivo development of polycystic kidney disease in thebpk-mutant mouse model. Bpk-mutant mice develop polycystic kidneydisease (PKD) starting at embryogenesis due to a point mutation in thegene encoding bicaudal C. All nephron segments are affected, and mostbpk-mutant mice die between postnatal days 24-30 due to severelyenlarged cystic kidneys and renal failure.

All mice were genotyped by PCR prior to treatment. Wildtype (Wt) andbpk-mutant (bpk) mice were then segregated into the following threegroups: no treatment (n=5 Wt, 2 bpk); vehicle treatment (n=5 Wt, 3 bpk);and EC0371 treatment (n=4 Wt, 4 bpk).

EC0371 was prepared by reconstitution in sterile PBS to a concentrationof 1 mM, then diluted 1:5 for a final concentration of 0.2 mM (2nmol/μl). Mice were injected (i.p.) daily with either EC0371 (3μmol/kg), vehicle (PBS), or received no injection, from postnatal day 7to day 21. On day 21, whole body weight was recorded and blood wascollected. Mice were then sacrificed and the kidneys, liver, spleen, andthymus were removed and weighed. EC0371 treatment of bpk-mutant mice wasfound to significantly improve the PKD phenotype as measured by kidneysize (FIG. 8), and proportion of kidney(s) to whole body weight (FIGS. 9and 10).

1. A method for diagnosing a kidney disease state, said method comprising the steps of: administering to a patient a composition comprising a conjugate or complex of the general formula V-L-D where the group V comprises a vitamin receptor binding ligand that binds to kidney cells and the group D comprises a diagnostic marker; and diagnosing the kidney disease state. 2-11. (canceled)
 12. A method for treating a kidney disease state, said method comprising the steps of: administering to a patient suffering from the disease state an effective amount of a composition comprising a conjugate or complex of the general formula V-L-D where the group V comprises a vitamin receptor binding ligand that binds to kidney cells and the group D comprises an antigen, a cytotoxin, or a cell growth inhibitor; and eliminating the disease state.
 13. The method of claim 12 wherein V comprises a folate.
 14. (canceled)
 15. The method of claim 12 wherein the group D comprises an antigen.
 16. The method of claim 13 wherein the group D comprises an antigen.
 17. The method of claim 12 wherein the group D comprises a cytotoxin.
 18. The method of claim 17 wherein the group D further comprises a liposome.
 19. The method of claim 13 wherein the group D comprises a cytotoxin.
 20. The method of claim 19 wherein the group D further comprises a liposome.
 21. The method of claim 12 wherein the group D comprises a cell growth inhibitor. 22-23. (canceled)
 24. The method of claim 13 wherein the group D comprises a cell growth inhibitor. 25-26. (canceled)
 27. The method of claim 21 wherein the cell growth inhibitor is rapamycin.
 28. The method of claim 24 wherein the cell growth inhibitor is rapamycin.
 29. A compound of the formula V-L-D, wherein V is a folate receptor binding ligand, L is an optional linker, and D is a cell-growth inhibitor.
 30. The compound of claim 29 wherein V-L-D has the following formula:

where L is as defined herein, and L is connected to the rapamycin or analog or derivative thereof at either of (O*), and the other of (O*) is substituted with R, wherein R is hydrogen or —CO(CR³R⁴)_(b)(CR⁵R⁶)_(d)CR⁷R⁸R⁹; where R³ and R⁴ are each, independently, hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, trifluoromethyl, or F; R⁵ and R⁶ are each, independently, hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, (CR³R⁴)_(f)OH, CF₃, F, or CO₂R¹¹; R⁷ is hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, (CR³R⁴)₁₀H, CF₃, F, or CO₂R¹¹; R⁸ and R⁹ are each, independently, hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, (CR³R⁴)_(f)OH, CF₃, F, or CO₂R¹¹; R¹¹ is hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of 2-7 carbon atoms, or phenylalkyl of 7-10 carbon atoms; b=0-6; d=0-6; and f=0-6.
 31. (canceled)
 32. The compound of claim 29 wherein the cell growth inhibitor is an epidermal growth factor receptor kinase inhibitor.
 33. The compound of claim 29 wherein the cell growth inhibitor is an inhibitor of mTOR.
 34. The compound of claim 29 wherein the cell growth inhibitor is a rapamycin.
 35. The compound of claim 29 wherein the folate receptor binding ligand is a folate.
 36. The compound of claim 29 wherein the linker is a peptide comprising one or more amino acids selected from the group consisting of cysteine, aspartic acid, and arginine, where the amino acid can be either the D or the L configuration in each instance. 